The control and treatment of bacterial infections in humans have been largely dependent on the use of antibiotics for the last five decades. However, the increasing incidence of antibiotic resistant bacteria demands the development of novel antibiotics. Comprehensive knowledge of the bacterial genes that are essential for in vitro growth and in vivo survival is crucial to identify promising targets for the development of effective antibiotics. The goal of this project is to develop experimental approaches for genome-wide identification of bacterial genes that are essential for in vitro growth or in vivo survival using S. enterica serovar Enteritidis (S. enteritidis) as a model organism. S. enteritidis is one of the most common serotypes of Salmonella causing food borne illness in the United States, posing a unique concern for public safety by routinely contaminating chicken shell eggs. We recently developed a method, termed "transposon sequence tag profiling" (TSP), for quantitative cataloguing of transposon insertions in a complex pool of mutants. The feasibility of TSP in functional characterization of a bacterial genome was demonstrated by screening a pool of approximately 500 S. enteritidis Tn5 mutants for attenuation during chicken infection. Two specific aims are proposed to accomplish the goal. We will use experimental approaches based on TSP to conduct genome-wide identification of the essential genes that are not dispensable for in vitro growth in Specific Aim 1 and the virulence genes that are required for in vivo survival during mouse infection in Specific Aim 2. The essential genes and the virulence genes in S. enteritidis that will be identified in this project should provide insights into the mechanisms of both in vitro and in vivo survival of this important food borne pathogen. These experimental approaches should find broad applications to other diverse bacterial species, which will contribute to the development of effective measures to control bacterial pathogens.